Lysosomal storage disorders (LSDs) are a group of approximately fifty inherited metabolic diseases that result from cellular deficiencies of a specific lysosomal enzyme, receptor target, activator protein, membrane protein, or transporter that leads to pathogenic accumulation of substances in lysosomes, causing accumulation of the substrate, resulting in deterioration of cellular and tissue function. Wilcox, W. R., Lysosomal storage disorders: the need for better pediatric recognition and comprehensive care. J Pediatr. 2004 May; 144(5 Suppl):S3-14. Lysosomal storage disorders occur in approximately 1 in 5,000 live births and display considerable clinical and biochemical heterogeneity. The majority of lysosomal storage disorders are inherited as autosomal recessive conditions although two examples of X-linked are MPS II and Fabry disease.
The extent and severity of the lysosomal storage disorder depend on the type and amount of substrate that accumulates, but almost all disorders are progressive. Most disorders have both central nervous system and systemic manifestations, whereas some affect either just the central nervous system or tissues outside the nervous system. Many patients with lysosomal storage disorders die in infancy or childhood, and patients who survive to adulthood often have a decreased lifespan and significant morbidity (Wilcox, 2004). Table 1, below, is a summary of some of the lysosomal storage disorders listed by the lysosomal function that is affected.
TABLE 1Lysosomal storage disorders by affected lysosomal function(Wilcox, W. R., J Pediatr. 2004 May; 144(5 Suppl): S3-14)Lysosomal function affectedDisorderDefective metabolism ofMPS I-IX (Hurler, Scheie, Hunter, Sanfilippo, Morquio,glycosaminoglycansMaroteaux-Lamy, Sly diseases, hyaluronidasedeficiency)Defective degradation of glycanAspartylglucosaminuria, fucosidosis, mannosidosis,portion of glycoproteinsSchindler disease, sialidosis type IDefective degradation of glycogenPompe diseaseDefective degradation ofFabry disease, Farber disease, Gaucher disease (typessphingolipid components1-3) GM1-gangliosidosis, GM2-gangliosidoses (Tay-Sachs disease, Sandhoff disease, GM2 activatordisease), Krabbe disease, metachromaticleukodystrophy, Niemann-Pick disease (type A or B).Defective degradation ofPycnodysostosispolypeptidesDefective degradation or transportCeroid lipofuscinosis (multiple types with differentof cholesterol, cholesterol esters, ordefects, some not known yet), cholesterol ester storageother complex lipidsdisease, Niemann-Pick disease type C, Wolman diseaseMultiple deficiencies of lysosomalMultiple sulfatase, galactosialidosis, mucolipidosis typesenzymesII, IIITransport and trafficking defectsCystinosis, mucolipidosis IV, sialic acid storage disorder,chylomicron retention disease with Marinesco-Sjogrensyndrome, Hermansky-Pudlak syndrome (severalforms), Chediak-Higashi syndrome, and Danon diseaseUnknown defectsGeleophysic dysplasia, Marinesco-Sjogren syndrome
Gaucher disease is the most prevalent lysosomal storage disorder and results from the deficiency of glucocerebrosidase (GC; EC 3.2.1.45) in all tissues. This enzyme deficiency produces accumulation of glucosylceramide in lipid laden macrophages (called Gaucher cells) in the reticulendothelial system including liver, spleen, lung, and bone marrow. Gaucher disease has been categorized into three major phenotypes: type 1, normeuronopathic; type 2, acute neuronopathic, and type 3, subacute neuronopathic. The spectrum of illness severity for type 1 Gaucher disease is diverse, where children and adults can be asymptomatic or may have severely debilitating symptoms, including skeletal degeneration, anemia, thrombocytopenia and hepatosplenomegaly. Symptoms can present at any age, and although type 1 Gaucher disease is more common among the Ashkenazi Jewish population, it occurs in all ethnic groups. Type 2 (acute neuronopathic) Gaucher disease is rapidly progressive, where by six months of age, most type 2 infants have brainstem dysfunction, and succumb to complications such as respiratory arrest or aspiration pneumonia at 18-24 months of age. Type 3 patients develop neurological abnormalities at a later age than type 2 patients; most only develop a subtle horizontal saccadic eye movement defect. Systemic complications in types 1 and 3 Gaucher patients respond to enzyme therapy.
Enzyme replacement therapy (ERT) for Gaucher disease was first approved by the FDA in 1991. Long-term ERT does improve the organomegaly and blood counts in most Gaucher patients. However, the current formulation of IV administered GC enzyme does not alter the neurological deterioration in type 2 patients or significantly reverse skeletal complications. To overcome the current limitations of ERT for Gaucher disease we propose to apply a novel technique that uses orally administered microscopic yeast cell wall particles containing DNA comprised of sequences encoding human glucocerebrosidase to more efficiently deliver normal or modified GC enzyme to macrophages. In addition to improved delivery of GC enzyme to all tissues, we expect this innovative approach will provide a more efficient and specific uptake by macrophages in bone of the particles that deliver the DNA encoding the normal or modified GC. This approach can lead to greater improvement in skeletal complications that are not significantly reversed by current ERT.
Extracted yeast cell wall particles are readily available, biodegradable, substantially spherical particles about 2-4 μm in diameter. Preparation of extracted yeast cell wall particles is known in the art, and is described, for example in U.S. Pat. Nos. 4,992,540; 5,082,936; 5,028,703; 5,032,401; 5,322,841; 5,401,727; 5,504,079; 5,968,811; 6,444,448 B1; 6,476,003 B1; published U.S. applications 2003/0216346 A1, 2004/0014715 A1, and PCT published application WO 02/12348 A2. A form of extracted yeast cell wall particles, referred to as “whole glucan particles,” have been suggested as delivery vehicles, but have been limited either to release by simple diffusion of active ingredient from the particle or release of an agent chemically crosslinked to the whole glucan particle by biodegradation of the particle matrix. See U.S. Pat. Nos. 5,032,401 and 5,607,677.
Extracted yeast cell wall particles, primarily due to their beta-glucan content, are targeted to phagocytic cells, such as macrophages and cells of lymphoid tissue. The mucosal-associated lymphoid tissue (MALT) comprises all lymphoid cells in epithelia and in the lamina propria lying below the body's mucosal surfaces. The main sites of mucosal-associated lymphoid tissues are the gut-associated lymphoid tissues (GALT), and the bronchial-associated lymphoid tissues (BALT).
Another important component of the GI immune system is the M or microfold cell. M cells are a specific cell type in the intestinal epithelium over lymphoid follicles that endocytose a variety of protein and peptide antigens. Instead of digesting these proteins, M cells transport them into the underlying tissue, where they are taken up by local dendritic cells and macrophages.
M cells take up molecules and particles from the gut lumen by endocytosis or phagocytosis. This material is then transported through the interior of the cell in vesicles to the basal cell membrane, where it is released into the extracellular space. This process is known as transcytosis. At their basal surface, the cell membrane of M cells is extensively folded around underlying lymphocytes and antigen-presenting cells, which take up the transported material released from the M cells and process it for antigen presentation.
A study has shown that transcytosis of yeast particles (3.4+/−0.8 micron in diameter) by M cells of the Peyer's patches takes less than 1 hour (Beier, R., & Gebert, A., Kinetics of particle uptake in the domes of Peyer's patches, Am J. Physiol. 1998 July; 275(1 Pt 1):G130-7). Without significant phagocytosis by intraepithelial macrophages, the yeast particles migrate down to and across the basal lamina within 2.5-4 hours, where they quickly get phagocytosed and transported out of the Peyer's patch domes. M cells found in human nasopharyngeal lymphoid tissue (tonsils and adenoids) have been shown to be involved in the sampling of viruses that cause respiratory infections. Studies of an in vitro M cells model have shown uptake of fluorescently labeled microspheres (Fluospheres, 0.2 μm) and chitosan microparticles (0.2 μm) van der Lubben I. M., et al., Transport of chitosan microparticles for mucosal vaccine delivery in a human intestinal M-cell model, J Drug Target, 2002 September; 10(6):449-56. A lectin, Ulex europaeus agglutinin 1 (UEA1, specific for alpha-L-fucose residues) has been used to target either polystyrene microspheres (0.5 μm) or polymerized liposomes to M cells (0.2 μm) (Clark, M. A., et al., Targeting polymerised liposome vaccine carriers to intestinal M cells, Vaccine. 2001 Oct. 12;20(1-2):208-17). In vivo studies in mice have reported that poly-D,L-lactic acid (PDLLA) microspheres or gelatin microspheres (GM) can be efficiently taken up by macrophages and M cells. (Nakase, H., et al., Biodegradable microspheres targeting mucosal immune-regulating cells: new approach for treatment of inflammatory bowel disease, J Gastroenterol. 2003 March; 38 Suppl 15:59-62).
However, it has been reported that uptake of synthetic particulate delivery vehicles including poly(DL-lactide-co-glycolide) microparticles and liposomes is highly variable, and is determined by the physical properties of both particles and M cells. Clark, M. A., et al., Exploiting M cells for drug and vaccine delivery, Adv Drug Deliv Rev. 2001 Aug. 23;50(1-2):81-106. The same study reported that delivery may be enhanced by coating the particles or liposomes with reagents including appropriate lectins, microbial adhesins and immunoglobulins which selectively bind to M cell surfaces. See also, Florence, A. T., The oral absorption of micro- and nanoparticulates: neither exceptional nor unusual, Pharm Res. 1997 March;14(3):259-66.
Pathogen pattern recognition receptors (PRRs) recognize common structural and molecular motifs present on microbial surfaces and contribute to induction of innate immune responses. Mannose receptors and beta-glucan receptors in part participate in the recognition of fungal pathogens. The mannose receptor (MR), a carbohydrate-binding receptor expressed on subsets of macrophages, is considered one such PRR. Macrophages have receptors for both mannose and mannose-6-phosphate that can bind to and internalize molecules displaying these sugars. The molecules are internalized by endocytosis into a pre-lysosomal endosome. This internalization has been used to enhance entry of oligonucleotides into macrophages using bovine serum albumin modified with mannose-6-phosphate and linked to an oligodeoxynucleotide by a disulfide bridge to a modified 3′ end; see Bonfils, E., et al., Nucl. Acids Res. 1992 20, 4621-4629. see E. Bonfils, C. Mendes, A. C. Roche, M. Monsigny and P. Midoux, Bioconj. Chem., 3, 277-284 (1992). Macrophages also express beta-glucan receptors, including CR3 (Ross, G. D., J. A. Cain, B. L. Myones, S. L. Newman, and P. J. Lachmann. 1987. Specificity of membrane complement receptor type three (CR3) for β-glucans. Complement Inflamm. 4:61), dectin-1. (Brown, G. D. and S. Gordon. 2001. Immune recognition. A new receptor for β-glucans. Nature 413:36.), and lactosylceramide (Zimmerman J W, Lindermuth J, Fish P A, Palace G P, Stevenson T T, DeMong D E. A novel carbohydrate-glycosphinglipid interaction between a beta-(1-3)-glucan immunomodulator, PGG-glucan, and lactosylceramide of human leukocytes. J Biol. Chem. 1998 Aug. 21:273(34):22014-20.). The beta-glucan receptor, CR3 is predominantly expressed on monocytes, neutrophils and NK cells, whereas dectin-1 is predominantly expressed on the surface of cells of the macrophages. Lactosylceramide is found at high levels in M cells. Microglia can also express a beta-glucan receptor (Muller, C. D., et al. Functional beta-glucan receptor expression by a microglial cell line, Res Immunol. 1994 May; 145(4):267-75).
There is evidence for additive effects on phagocytosis of binding to both mannose and beta-glucan receptors. Giaimis et al. reported observations suggesting that phagocytosis of unopsonized heat-killed yeast (S. cerevisiae) by murine macrophage-like cell lines as well as murine peritoneal resident macrophages is mediated by both mannose and beta-glucan receptors. To achieve maximal phagocytosis of unopsonized heat-killed yeast, coexpression of both mannose and beta-glucan receptors is required (Giaimis, J., et al., Both mannose and beta-glucan receptors are involved in phagocytosis of unopsonized, heat-killed Saccharomyces cerevisiae by murine macrophages, J Leukoc Biol. 1993 December; 54(6):564-71).